HLA Class I Polymorphism Analysis. PA0 1. Introduction
The major histocompatibility complex (MHC) includes the human leukocyte antigens (HLA) gene complex which is located on the short arm of human chromosome six. These genes encode cell-surface proteins which regulate cell-cell interactions of the immune response. The various HLA Class I loci encode the heavy chain of the Class I molecules (44,000 dalton polypeptide), which associate with B-2 microglobulin. The different Class I dimers are expressed on the surface of essentially all human cell types. These molecules are involved in the presentation of endogenous peptides (i.e., virally-encoded proteins) by the expressing cells; this molecular complex is recognized on the target cells by cytotoxic T lymphocytes, in a self-restricted manner.
The HLA-A, -B and -C loci of the HLA Class I region exhibit an extraordinarily high degree of polymorphism. The WHO nomenclature committee for factors of the HLA system [Marsh and Bodmer, Immunogenetics, 31:131 (1990)]designated 25 alleles at the HLA-A (HLA-A,0101, A*0201, etc.), 32 alleles at the HLA-B, and 11 alleles at the HLA-C loci. Since this high degree of polymorphism relates to the function of the HLA molecules, much effort has gone into designing accurate and optimal methods for detecting the many possible alleles that may be carried at each locus by any given individual of the population. Products of the HLA genes were first identified by reactions of antisera. Serological techniques remain the primary, and in many cases the only, typing method for HLA antigens. The complement-dependent cytotoxicity (CDC) assay is the method most often used to define serologic specificities [Terasaki and McClelland, Nature, 204:998, (1964)]. The advantages of the CDC include the small volumes of antisera and target cells and the relatively short time required for the test.
One great disadvantage of serological typing is that alloantisera are not infrequently limited in number and volume, and often have complex reactivities, so that an extensive program is required to identify and obtain useful sera. Perhaps most importantly, however, many cellularly and/or biochemically defined polymorphisms, which in all likelihood have functional significance in bone marrow transplantation, cannot be detected by serological techniques [Anasetti et al., Hum. Immunol., 29:70 (1990)].
One-dimensional isoelectric focusing (1D-IEF) is very effective in identifying serologically undetectable variants or subtypes for the HLA-A and HLA-B antigens [Yang, Immunobiology of HLA, Vol. I, 332 (1989)]which have been implicated in the development of acute graft-versus-host disease and in graft rejection in bone marrow transplantation. In this technique, metabolically labeled cell lysates are immunoprecipitated using monoclonal antibodies to Class I antigens and the immunoprecipitates are desialated and subjected to isoelectricfocusing. The technique, however, is time consuming and cumbersome. Interpretation of the IEF patterns is dependent on prior knowledge of the serological definition, and certain HLA antigens show so many overlapping bands that IEF assignment is difficult and impractical.
It is believed that only about 30% of the existing Class I sequences are known as of today. This limited knowledge extraordinarily complicates the application and use of other molecular approaches for Class I HLA typing which are being used for Class II typing (i.e., oligotyping). Furthermore, because the distribution and nature of the sequence polymorphisms in the Class I genes is very different from that seen in Class II genes, oligotyping strategies for Class I genes may require the use of a very large number of oligonucleotides to type the alleles known at the present time and this number may become much larger as new allelic sequences are described. Application of a sequence-based typing technique to the analysis of HLA Class I polymorphism presented a series of problems additional to those associated with HLA Class II typing. These problems are: 1) the informative polymorphism at Class I loci spans 2 different exons (approximately 600 base-pairs), instead of 1 in Class II genes; 2) the HLA-A, -B and -C loci are believed to be far more polymorphic than Class II loci; 3) the different Class I loci are far more related to each other in terms of DNA and amino acid sequence than Class II loci are to each other. Thus, when the alleles at each locus are compared with each other, they resemble alleles at a single locus and, therefore, given that up to 6 different Class I genes may be expressed by a given individual (2 alleles at each locus) this increases the number of technical limitations to molecular typing imposed by this polymorphic system.
Accordingly, there is a need for a method to determine genomic information from such a highly polymorphic system as the HLA- Class I system that addresses the limitations imposed by previous methods. That is, a system that is capable of determining the nucleotide sequences of the genes carried by any given individual without the need to have previous knowledge of the individual's HLA type as defined by other methods.
The present invention provides a molecular approach for accurate HLA Class I sequence-based typing that is rapid, avoids the use of oligonucleotides specific for each known allele, requires the use of only a small number of oligonucleotide primers, does not require previous typing information, can readily detect new sequence variants unidentifiable with more conventional approaches, and is entirely automatable.